While much of current biology focuses on proteins, and their uses, it must not be forgotten that other molecules are extremely important in a number of biological contents. Lipids, and lipid containing molecules (e.g. glycolipids), have an important role as pharmaceutical agents, immunogens, and so forth.
Exemplary of the important lipid containing molecules is the class of glycolipids referred to as gangliosides. Gangliosides belong to a family of glycolipids referred to as glycosphingolipids. These glycosphingolipids contain at least one neutral sugar residue at the polar head group of a lipid molecule. Gangliosides are molecules which contain at least one sialic acid residue in this oligosaccharide head. These sialic acid residues give the head, and the glycolipid molecule itself, a net negative charge. For a discussion of gangliosides, and of glycolipids and their structures in general, attention is drawn to the well known textbooks, Lehninger, Biochemistry (Worth Publishers, 1981), pp. 287-295, especially 294-295. This material is incorporated by reference in its entirety. Exemplary of the important role of gangliosides in a therapeutic context is Mahadnik et al., "Gangliosides In Treatment of Neural Injury and Disease", Drug Development Res. 15: 337-360 (1988). See also U.S. Pat. No. 4,710,490 to Catsimpoolas et al. (gangliosides and angiogenesis); Mynard et al, U.S. Pat. No. 4,347,244 (GM1 and diarrhea); Horowitz, "Ganglioside (Cronassial) Therapy in Diabetic Neuropathy", Adv. Exp. Med & Biol 174: 593,600 (1984); Karpiatz et al., "Exogenous Gangliosides Enhance Recovery from CNS Injury", Adv. Exp. Med. & Biol. 174: 489-497 (1984), all of which are incorporated by reference. The Catsimpoolas et al. patent is of special relevance in that it sets forth in great detail, flow charts summarizing various protocols for securing lipid containing molecules, while Catsimpoolas et al used mammalian omentum as a source for their gangliosides and lipids, other tissue sources may be used, as is elaborated infra.
Lipids, unlike proteins, are not coded for by genes. Thus, one desiring large amounts of lipid containing molecules does not have the resources afforded by recombinant genetics available to him or her. Securing large amounts of lipid containing material forces the investigator to confront the problems and pitfalls of natural product chemistry.
Most lipid containing materials are secured after a rich source of the lipid material of interest is identified, and an extraction protocol developed. Generally, this requires some treatment of the source material, followed by contact with a solvent. The lipid of interest is then separated from the solvent.
The methodology is never as simple as presented, however. To begin with, "rich source" is very misleading. For many lipid containing materials, there is no "rich source", as the total yield can range between nanograms and micrograms. Further, within a generic group of materials, variation from sample to sample should be expected. Also, extraction protocols, even in their most defined formats, are frequently very harsh on the target molecule or molecules. Frequently, there is a loss of product which can be attributed to the purification protocol itself. Another issue is the fact that many lipid molecules demonstrate extremely similar structures and properties in solution, so it is very difficult to resolve individual species following extraction.
The foregoing are simply exemplary of the problems in the field, and should not be taken to be comprehensive of all issues as faced by the artisan. Some headway has been made however, and this is the subject of the invention.
Veterinary medicine is aware of a condition referred to as ovine GM1 gangliosidosis, as reported by, e.g., Ahern-Riddel et al, Biochem. Genet. 26: 733-746 (1988); Ahern-Rindell et al, Somat, Cell & Molecular Genet. 15(6): 525-533 (1989); Murane, et al., Am. J. Pathol. 134:263-270 (1989); Prieur et al., J. Hered. 81: 245-249 (1988), Prieur et al., Am. J. Pathol. 139(6): 1511-1513 (1991); all of which are incorporated by reference in their entirety. The disease, which affects lambs, is characterized by a profound deficiency in acid .beta.-galactosidase activity, and a partial deficiency in .alpha.-neuraminidase activity. The condition does have a parallel in humans, i.e., human GM1 gangliosidosis, although the decrease in a neuraminidase levels has not been found in humans. Analysis of the neural tissue of lambs afflicted with GM1 gangliosidosis has revealed that these tissues contain surprising large amounts of lipid containing molecules, especially glycolipids such as gangliosides. The levels observed are several orders of magnitude greater than the vanishingly small amounts found in other tissue sources.
Hence, it is an object of the invention to provide a method for obtaining enhanced yields of lipid containing molecules, via extraction of neural tissue of animals, e.g., ovine animals, suffering from GM1 gangliosidosis.
How this, and other aspects of the invention are achieved will be seen from the disclosure which follows.